Mythimna separata, the oriental armyworm, is a lepidopteran pest that threatens cereal crops. In the current study, two strains (XSBN200920 and JHML200710) of entomopathogenic fungus Metarhizium rileyi were tested for virulence against oriental armyworms. When treated with spore suspensions of both strains at a concentration of 1.0 × 10 spores/mL, the 3rd instar larvae's survival rate was considerably different (P < 0.01). The median lethal time of the insects exposed to XSBN200920 was about 3 d longer than that of JHML200710. The results of 16S ribosomal RNA sequencing showed that Chao1 richness in the JHML200710 treatment group was significantly decreased compared with the CK ( 0.02% Tween 80). The dominant gut bacteria species at the phylum level were Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidota in the three groups. The CK group had a much higher associated abundance of cyanobacteria than the other two fungal treatment groups. Sixteen genera revealed significant variations in the gut bacteria of the insects at the genus level. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional gene and enzyme analysis showed that when compared with the CK group, the XSBN200920 treatment group showed a significant reduction in six aspects, including betalain biosynthesis, spliceosome, and neuroactive ligand-receptor interaction. These findings suggested that healthy and fungus-infected insects' intestinal microbial community structure differed significantly. And the virulence of M. rileyi is closely linked to its ability to alter the structure of the intestinal microbiome of insects. The results offer a starting point for examining the relationship between the gut microbial diversity of oriental armyworms and variations in the virulence of pathogenic fungi.